Stable trapping of multiple proteins at physiological conditions using nanoscale chambers with macromolecular gates

The possibility to detect and analyze single or few biological molecules is very important for understanding interactions and reaction mechanisms. Ideally, the molecules should be confined to a nanoscale volume so that the observation time by optical methods can be extended. However, it has proven difficult to develop reliable, non-invasive trapping techniques for biomolecules under physiological conditions. Here we present a platform for long-term tether-free (solution phase) trapping of proteins without exposing them to any field gradient forces. We show that a responsive polymer brush can make solid state nanopores switch between a fully open and a fully closed state with respect to proteins, while always allowing the passage of solvent, ions and small molecules. This makes it possible to trap a very high number of proteins (500-1000) inside nanoscale chambers as small as one attoliter, reaching concentrations up to 60 gL−1. Our method is fully compatible with parallelization by imaging arrays of nanochambers. Additionally, we show that enzymatic cascade reactions can be performed with multiple native enzymes under full nanoscale confinement and steady supply of reactants. This platform will greatly extend the possibilities to optically analyze interactions involving multiple proteins, such as the dynamics of oligomerization events.

running buffer has pH ≈ 6. No differences in adsorption and desorption behavior can be seen.
The dissipation signals were negligible (~0.3 × 10 −6 ). Interestingly, we noticed some differences for BSA if it was instead labelled with fluorescein. This version seemed to adsorb in higher amounts, though it was also fully desorbed at high pH. Furthermore, we saw indications that for native BSA, the adsorption and desorption behavior was dependent on factors such as batch, product number or supplier. This could potentially be related to impurities, in particular other proteins. We emphasize that care must be taken to characterize the adsorption and desorption behavior of each protein (preferably using SPR or QCM) before trapping in nanochambers. images. The first image shows the intensity before any fluorescent protein has been introduced.

Time [min]
The first step is a control where proteins are introduced at room temperature, when the gates should be closed. The last step is a control where only the pH is changed back to 6.0. Note that spots appear primarily in steps 3 and 4. All images were obtained without proteins in the bulk solution and at room temperature.  NAD + in solution is reduced by alcohol dehydrogenase (ADH) into NADH, which is detected by its absorbance at 340 nm. No significant increase was measured at 430 nm. This "cofactor transport" system proves that NAD + diffuses into the nanochambers and that NADH diffuses out. (If NADH would be stuck inside the nanochambers the reaction would stop and the absorbance change would be negligible.) Additionally, by including other enzymes in the liquid environment outside the chambers, a "cofactor cycling" system was created which quickly eliminates the NADH formed. Here GOX and glucose produces H2O2, while horseradish peroxidase (HRP) and ferricyanide are additional redox mediators since H2O2 alone does not oxidize NADH. 4 The effect from HRP without ferricyanide was noticeable, as expected, 5

Supplementary Notes 1
Reason for the small negative signal upon pH increase using nanoplasmonic sensing.
For very thin brushes, the negative signal when the pH is increased is of course mainly due to molecules leaving the chambers (Supplementary Figure 7a). However, even when the brushes were closing the chambers at RT (no detectable signal from BSA), a small negative signal was always seen when desorbing the proteins from the nanochamber walls (Figure 4a in main text).
Yet fluorescence data did not show any corresponding signal decrease at the pH increase step (Figure 4b), which suggests that this is an effect of the plasmonic readout method. We attribute these observations to the inhomogeneous near field distribution in the nanochambers. 8 When the proteins leave the chamber walls they are likely, on average, located in regions where the sensitivity to refractive index changes is lower. This view is consistent with the fact that the signal decrease was not instant but followed the desorption kinetics of BSA very well.

Supplementary Notes 2
Origin of the fluorescence signal from nanochambers (compared to planar gold) at RT.
In Figure 4b, a small but significant fluorescence intensity difference is observed when comparing nanochambers and planar gold after exposure to BSA at RT and pH 6. Since the amount of exposed gold is the same on both regions, it is tempting to attribute this signal to a fraction of nanochambers that are open at RT. However, the small intensity remains even after raising the pH when the chambers are open, where one would expect it to go away due to desorption. Although minor, the signal calls for an additional explanation, which we believe is a combination of the inability of the polymer brush to fully block protein adsorption to gold and differences in transmission through the two surface regions. Due to the high sensitivity of fluorescence detection compared to label-free plasmonic sensing, it would be surprising if there would be absolutely no fluorescence at all after exposing PNIPAM-modified gold to labelled BSA. Indeed, some proteins could be detected in between individual nanochambers on the sparse arrays (Figure 5a in main text), potentially associated with a few defects from the nanofabrication process (e.g. colloids that were not removed). Furthermore, the extinction of the dense nanochamber array is generally lower than for a gold film of the same thickness. 9 (This is not true at the resonance peak, but the fluorophore emits at shorter wavelengths.) Since the intensity is measured through the glass support, emitted light will pass through the gold and the plasmonic activity will therefore influence the intensity that reaches the detector from the nanochamber region. (The effect was clearly observed when there were fluorescent molecules in the liquid bulk, in which case a higher intensity went through the nanochamber region.) Thus, small amounts of protein adsorption on gold, potentially due to minor defects in the polymer coating, can explain the higher intensity measured through the nanowells.

Supplementary Notes 3
Reasons for (a small fraction of) malfunctioning chambers.
As mentioned in the main text, individual nanochambers not containing proteins after the pH increase step may be the result of the brush being (on average) either too high or too low ( Figure   5a in main text). We could identify cases in the single nanochamber experiments where the brush was indeed much too thick or too thin by recording fluorescence from individual nanochambers during all the different steps in the trapping. Following Supplementary Figure   9, if the brush was generally too thick there were very few fluorescent spots correlating with nanowells in step III. Similarly, if the brush was generally too thin, many fluorescent spots correlating with nanowells appeared already in step II and disappeared in step IV. This is essentially the same findings as obtained from plasmonic dense arrays in Supplementary Figure   7. However, even when the polymerization time was around the optimum for creating the right brush thickness, we could still identify some nanochambers that did not capture proteins as intended. In these cases the results did not indicate a too thick or a too thin brush, but rather a mixture of both. This can be partly attributed to the size distribution of the nanochamber openings, but it is quite narrow with almost all the aperture diameters within ±10 nm. 9 Therefore, we also believe that non-uniformity of the PNIPAM brush is an important effect, i.e. that the thickness varies over the sample area even if the average value is indeed suitable for trapping. This is consistent with the liquid-phase AFM data (Supplementary Figure 1) which does not show a very smooth surface in between the nanochambers. In summary, although most nanochambers work as intended when the ATRP is performed carefully, future improvements should focus not only on fine control of the average brush thickness, but also its uniformity by varying factors such as reactant/catalyst concentrations and solvent composition.

Supplementary Notes 4
Protein denaturation and the importance of silanization.
As mentioned in the main text, BSA is known to not undergo any irreversible changes in structure due to adsorption to and desorption from silica through pH changes. This is likely the case for many proteins because, in general, protein denaturation occurs on hydrophobic surfaces where hydrophobic interactions with their interior makes them unfold. On silica, tunable electrostatic interactions can be used to cause adsorption that can be reversible with pH (or potentially salt) while the protein remains folded. However, it cannot be assumed that this approach will be non-invasive for all water-soluble proteins. Therefore, the possibility to use silanization of the nanochamber interior surface (Supplementary Figure 10) is very important.
Proteins are even less likely to denature if adsorbed on an organic film. The broad range of silanization chemistries available, including recent improvements with respect to monolayer uniformity and click chemistry for further modification, 10 opens up for many possibilities to ensure that proteins are adsorbed and released in a gentle manner. It is even possible to release proteins adsorbed to the walls by conventional immobilization protocols, such as His-tags. The eluents used are normally small molecules (imidazole for His-tags) which can diffuse through the brush barrier. The salt content can be changed temporarily during such a desorption process.
With such an approach, there is not even a need to change pH.